Project Summary Ehrlichia chaffeensis (E. ch.) is a gram-negative, obligately intracellular bacterium and causative agent of the most prevalent life-threatening tick-borne disease in the United States, human monocytic ehrlichiosis (HME). Wnt signaling is a conserved eukaryotic signal cascade comprising canonical and noncanonical pathways that regulate events including cell fate, development, and cell polarity, as well as innate immunity-associated events such as autophagy, cytokine expression, and phagocytosis. Our laboratory has shown that during infection, E. ch. activates conserved eukaryotic signaling pathways including both canonical and noncanonical Wnt signaling. Wnt signaling enhances E. ch. intracellular survival by driving bacterial uptake and inhibiting fusion of the ehrlichial replicative vacuole with the lysosome. Although these studies have identified Wnt pathway activation as a virulence strategy for E. ch., identification of an activating event for the observed phenomena remains a critical gap in knowledge. Under normal physiological conditions, Wnt signaling- dependent phagocytosis is initiated through the binding of a Wnt ligand to one of 10 Frizzled (Fzd). Our preliminary data demonstrates that E. ch. surface protein TRP120 directly binds a Fzd, possesses homology with the conserved family of Wnt proteins, and can stimulate activation of the Wnt transcription factor ?- catenin. We have also shown that inhibition of Wnt signaling blocks ehrlichial entry, indicating E. ch. effectively establishes infection through activation of Wnt-dependent phagocytosis. The long-term goal of this project is to utilize E. ch. manipulation of monocyte Wnt signaling as a model to study the therapeutic potential of harnessing Wnt signaling during human intracellular bacterial infection. The objective of this proposal is to define the bacterial ligand and eukaryotic receptor determinants of E. ch. effector-driven activation of Wnt signaling and entry into monocytes. We hypothesize that ehrlichial TRPs are Wnt ligand mimetics that signal through Wnt pathway receptor-coreceptor pairs for activation of canonical and noncanonical Wnt signaling to enhance bacterial host cell entry and intracellular survival. In specific aim 1, we will investigate ehrlichial TRP Wnt ligand mimetic activation of canonical and noncanonical Wnt signaling. In specific aim 2, we will define the role of Wnt pathway receptors and coreceptors in ehrlichial TRP-driven Wnt signaling activation during infection. This research will provide insight to evolutionarily conserved eukaryotic pathways that pathogens have evolved to utilize for cell invasion and intracellular growth. Our approach to identifying a level at which Wnt signaling can be hijacked by intracellular pathogens will provide mechanisms for previously observed phenomena as well as potential antimicrobial therapeutic targets